Technical Abstract:
Variation in aster yellows phytoplasma (Candidatus Phytoplasma asteris) titer in its insect vector, Macrosteles quadrilineatus
The aster yellows phytoplasma (AYp) is transmitted by the aster leafhopper (ALH), or Macrosteles quadrilineatus, in a persistent and propagative manner. To study AYp replication and examine the variability of AYp titer in individual ALHs, we developed a quantitative real-time PCR (qPCR) assay to measure AYp concentration in insect DNA extracts. Absolute quantification of AYp DNA was achieved by comparing the amplification of unknown amounts of an AYp target gene sequence, elongation factor TU (Tuf), from whole insect DNA extractions to the amplification of a dilution series containing known starting quantities of the Tuf sequence cloned into a plasmid. The abilities and limitations of this method were assessed by conducting a time course experiment that varied the incubation time of AYp in the ALH from 4 to 25 days following a 48 hour acquisition access period (AAP) on an AYp source plant. AYp concentration was measured in 73 ALHs and the average titer, expressed as log (copies/ng DNA), ranged from 2.59 (±0.68) to 4.46 (±1.08) occurring at 4 and 13 days post acquisition in both male and female insects. In general, AYp titer increased over time and became asymptotic after 10 days at a concentration of 4.32 copies per ng DNA. AYp concentration in an ALH control group not given access to source plant averaged 0.75 (±0.09). AYp titers measured using the Tuf target sequence were significantly correlated (r = 0.99; p < 0.001) to titers measured using the AYp gene target, lysyl-tRNA synthetase (LystS). This new method will enable us to examine the biological factors governing AYp replication in the ALH and examine if AYp population size is associated with the frequency of transmission.